Center for Innovations in Medicine Immunosignatures: a Platform Technology for Diagnosis and Discovery RUSNANO Stephen Albert Johnston Center for Innovations in Medicine HealthTell Russian American Anti Cancer Center 1
Current Center for Innovations In Medicine Projects OBJECTIVE INVENTION COMPANY Health Monitoring/ Immunosignatures HealthTell, Inc Early Diagnosis Universal Preventative Frameshift Antigens Calviri, LLC Cancer Vaccine NextGen Antibiotics, Synbodies Anti-Virals 2
Forbes Magazine, 1/19/2012 U.S. Healthcare Hits $3 Trillion National Healthcare Expenditure – or NHE. … NHE for 2012 is probably closer to $2.7 trillion but there’s this nagging bookkeeping accrual of about $300 billion where we (narrowly) avoided those darn pesky SGR cuts to Medicare. … That puts the real NHE at about $3 trillion for 2012 (+ about 4% for each year forward – as far as the eye can see). As one economist said – we don’t have a debt problem in this country – we have a healthcare problem. http://www.forbes.com/sites/danmunro/2012/01/19/u-s-healthcare-hits-3-trillion/ $3 trillion is ~19% of the GDP for the US 3
Cancer Deaths Developed Worldwide (high-income/ Developing & industrialized): under- 29% of cancer developed deaths countries: 2,054,897 71% of cancer deaths/yr. deaths 5,053,872 deaths/yr . 7,108,769 total cancer Source : World Health Statistics 2006 , published by the World deaths/yr. Health Organization (WHO). WHO.
WHO: Imminent global cancer 'disaster'
“The total economic impact of premature death and disability from cancer worldwide was $895 billion in 2008.”
Anne Weston, picture of “ How to Cure Cancer ” , Time magazine web, June,2013
“When the stars come together cancer doesn ’t stand a chance”
Positron Emission Therapy Development: $200B (1000 centers) Cost/Year: $50B Cost/Trtment ~$30,000 Physicists 10,000
Personalized Medicine 1. Tumor DNA and/or RNA from ONE Individual is Sequenced 2. Analysis of Sequence Indicates the Right Drug to Use 3. Treatments often >$100,000 US
Post -Symptomatic Medicine To Pre-Symptomatic Health Per capita health expenditure ~$8000 Median adjusted gross income in 2007 ~$33,000* Median federal taxes per capita in 2007 ~$1,000 Total Medicare expenditures in 2004 ~$3B 2009 GNP $14.7T Medicare expenditure per capita ~$1000 2009 Health Care Costs $2.5T Graphs from “Trends in Health Care Costs and Spening, Kaiser Family Foundation, March 2009 http://www.kff.org/insurance/upload/7692_02.pdf; *gross income and tax data from the Tax Foundation http://www.taxfoundation.org/news/show/250.html
Transition from Post- to Pre-Symptomatic Medicine Requires System to Continuously Monitor Health of Well People Specifications Required: - Comprehensive - Sensitive – Early Detection - Simple - Inexpensive - Specificity – What is Wrong?
Can Not Do Early Detection of Disease Blood Dilution Problem 10 4 Improvement in Detection Needed Sci Transl Med 3 , 109ra116 (2011); Sharon S. Hori , et al. Detection Strategies and Limitations Mathematical Model Identifies Blood Biomarker - Based Early Cancer
The Immune System Detects and Amplifies Signal • 10 8 to 10 9 antibodies exist in serum • A single reactive B cell encounters antigen and is activated • Produces 5,000 to 20,000 antibodies per minute • Divides every 70 hours • Signal is amplified ~10 11 times in one week
Immunosignatures: A universal, simple and cheap platform for disease diagnosis Peptides Disease Normal Subjects CIM10K: 10,000 non-natural sequence peptides Sykes et al. 2013 Trends Biotechnol. 31(1):45-51
Toward a World Without Patients
Center for Innovations in Medicine Immunosignature Process Array of 10K-350K, Addressable, Non-Natural Sequence Space Peptides 17
Center for Innovations in Medicine Immunosignature Process Add diluted blood 18
Immunosignature Process Wash 19
Immunosignature Process • One array for all samples, human and nonhuman • Very small quantity of blood required • Scalability and low cost array fabrication Wash 20
Problem: How to Display Ab Diversity Antibody Diversity 10 9 Different Ab/person 10 19 Peptide Sequence Space In 3 x 10 5 peptides
Nature Does Not Always Know Best - Life occupies an infinitesimally small part of potential sequence space - Therefore there are many other sequences that could be useful - Peptides on array are chosen to evenly sample random sequence space(3.5x10 5 / 10 21 possibilities) Consequences: Same set of peptides can be used for any diagnosis Super-fine resolution of antibody diversity
Monoclonal Antibodies Bind Distinct Patterns on the Array Stafford et al. Mol Cell Proteomics 2012. 11(4):M111.011593
Information from Each Feature Intensity (#Ab/spot) Amino Acid Sequence (Repeating Motifs) Feature Isotypes On Array IgG (4) IgM IgE IgA
Peptide Microarray Vs ELISA Sera Secondary 1:1,634,800 Alone
Identifying The Immunosignature 10,000 Peptides p < 1x10 -6 & Fold Change Control ~ 100 informative peptides Disease Train a Machine Learning Algorithm
Performance is Tested on a Second Group ROC Curve 1 0.9 0.8 0.7 True Postive Fraction 0.6 0.5 0.4 0.3 0.2 0.1 0 0 0.2 0.4 0.6 0.8 1 1.2 False Postitve Fraction Disease Control
Features of Immunosignatures Same chip used for all diseases, all species Detects all antibodies: sugars, non-linear, modifications Historical sera samples work No sample preparation 10-100x more sensitive than ELISA
One Chip, Many Tests West Nile Type 1 Diabetes Breast Cancer
Dry Blood Works as Well as Fresh Blood Dried vs. Undried Whole Blood Immunosignature Correlations 0 Week Dried vs. 1 Week Dried vs. 3 Week Dried vs. Undried Whole Blood Undried Whole Blood Undried Whole Blood Storage conditions: 25C R 2 = 0.919 R 2 = 0.916 R 2 = 0.843 30
Platform 1: Printed Arrays of 10,000 Peptides 17 amino acids long, random sequence, and all amino acids except C are used. Two copies of the library are printed on a glass slide (~1200 peptides/cm 2 ). Mass spectra available for all peptides spotted on the arrays. 31
• UV photolithography • All chemistry performed using coater/developer • Large number of cycles makes the process challenging 32 32
1.2 Stepwise Yield Analysis Antibody Probe Ab1 DM1A Ab8 1 A 0.998 V 0.999 0.8 P 0.999 0.6 L 0.992 G 0.991 0.4 Y 0.992 F 0.998 0.2 S 0.998 0 DM1A N 0.998 DM1A DM1A Ab8 Ab1 Ab8 Ab1 Ab8 Ab1 Epitope Q 0.986 K 0.972 D 0.997 E 0.998 H 0.711 G-Peg-GSGPQL-Tmpp G-Peg-GSGLAN-Tmpp His oxidized to Asn G-Peg-GSGFGS-Tmpp G-Peg-GSGFYY-Tmpp Material in this presentation contains ASU and HealthTell Intellectual Property and is Confidential
Cost per chip Vs volume • Cost analysis includes • Labor cost – Technicians need to run the Cost per chip tools $350 • Yield & QA cost – Labor + reagents – Each batch sample set is $300 9,033, $290 analyzed • Chemical/biochemical reagents $250 – Surface prep – Litho & resist steps Cost per chip – Amino-acid coupling $200 • Materials – Wafer $150 – Mask set • Facility cost 25,135, $108 – Rent, unitlity, & chemical $100 disposal • 45,164, $63 Tool maintenance $50 • Packaging 90,327, $35 180,655, $21 361,309, $13 $0 5,000 50,000 500,000 Number of chip
Array Production Breakdown: Wafer to Individual Spot Multiple 24 Arrays Features Single Completed Single Array Diced Slide Feature Wafer 350K peptides/array ~50-Fold Improvement Over CIM10K Printed Arrays
OneTest ™ Comprehensive Health Monitoring Please Do Not Distribute 36
Center for Innovations in Medicine Valley Fever (Coccidiomycosis) • About 30,000 reported cases annually • Particularly prevalent the Sonoran desert • While most cases mild, it can be life threatening • Flu-like symptoms Coccidioides immitis spherule with endospores .
Classic Train/Test Example: Valley Fever Normal Infected • 10,000 peptides on original array • 120 patients screened and analyzed • 100 most informative peptides selected and resynthesized John Galgiani Univ. of Arizona • Diagnostic array printed 38
Outperforms Existing Diagnostic Patients with Valley Fever Patients that did not have Valley Fever • 90 blinded samples from patients presenting at the clinic • Zero false positives (100% specificity) • Zero false negatives (100% sensitivity) All Patients with Valley Fever Presented with Zero CF Titers, but were later shown to have the disease 39
Breast Cancer Test/Train using geographically distinct cohorts Healthy Breast Cancer
Immunosignaturing Brain Tumors Collaborator: Adrienne C. Scheck, BNI 100% accurate detection training and testing on samples taken years apart using printed arrays Not only can immunosignaturing detect the brain tumor, it can distinguish accurately between the common types of brain tumors 41
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